2-aza-2-desamino analogues of 5,8-dideazafolic acid

ABSTRACT

2-Aza-2-desamino analogues of 5,8-dideazafolic acid, which analogues have the formula  &lt;IMAGE&gt;  wherein X is an aryl or heteroaryl moiety; R1 is H, C1-C4 alkyl, C3-C4 alkenyl, or C3-C4 alkynyl; and R2 is OH, an L- alpha -amino acid, or a peptide comprising L- alpha -amino acids.

This invention was made in the course of work supported in part by theU.S. Government, which therefore has certain rights in the invention.

The field of the invention is folic acid analogues.

BACKGROUND OF THE INVENTION

Methotrexate (MTX) is a classical antifolate drug used for many years inthe treatment of human cancers. This drug exerts its primary biologicaleffect by inhibiting dihydrofolate reductase (DHFR), an enzyme which isresponsible for the terminal step in converting the vitamin folic acidinto a reduced form that actively participates in several crucialmetabolic pathways within cells. The usefulness of MTX as a cancerchemotherapeutic, however, is often limited by intrinsic or acquiredresistance of tumor cells to its effects, so that alternativeantifolates effective against MTX-resistant tumors have been sought.

The potential therapeutic significance of folic acid analogues targetedagainst thymidylate synthase (TS) as opposed to DHFR was predicted morethan 20 years ago by Borsa et al. (Cancer Res. 29:737, 1969). Shortlythereafter, the potent biological activity of 5,8-dideazafolic acid (1)was reported (Hutchison et al., Proc. Amer. Assoc. Cancer Res. 10:80,1969; Bird et al., J. Mol. Pharmacol. 6:573, 1970). This led to anextensive program of synthesis of quinazoline analogues, and to theeventual selection of the N¹⁰ propargyl derivative 2 (CB3717, PDDF) as asuitable candidate for biochemical and clinical evaluation. While thiscompound has many desirable evaluation. While this compound has manydesirable pharmacological characteristics, such as the ability to entercells by a transport mechanism distinct from that of reduced folates andMTX, its clinical usefulness is hampered by hepatic and renaltoxicities, which are due in part to low solubility at physiologic pH.

The toxicity problems encountered during clinical trials with CB3717prompted a vigorous search for more soluble congeners, culminating inthe discovery of a second-generation family of folate-based TSinhibitors, of which the prototypical example was2-desamino-5,8-dideazafolic acid (3) and its N¹⁰ -methyl (4) and N¹⁰-propargyl (5) analogues (Marsham et al., J. Med. Chem. 32:569, 1989;Jones et al., J. Med. Chem. 32:847, 1989). Also active were thecorresponding 2-desamino-2-methyl analogues 6-8 and some of theircongeners. ##STR2##

Biochemical studies with compounds 3-8 revealed that while replacementof the NH₂ group at C² by H or methyl generally results in weakerbinding to purified TS in vitro, it also results in increased inhibitorypotency against intact cells in culture (Jackman et al., Cancer Res.50:5212, 1990; Jones et al.; Hughes et al., J. Med. Chem. observationsare explained by the findings that (i) deletion of the 2-amino groupdoes not diminish the analog's substrate activity forfolylpoly-glutamate synthetase (FPGS), and (ii) polyglutamylation of the2-desamino compounds increases binding to TS by as much as 100-fold(Moran et al., Mol. Pharmacol 36:7836, 1989; Jackman et al.). On thisbasis, a large number of congeners modified at C², N¹⁰, and the aroylmoiety were synthesized to permit a detailed structure-activity analysisof 2-desamino-5,8-dideazafolates relative to the parent 2-aminocompounds. The thiophene analogue 9 (ICI-D1694), with a methyl group atC² and a methylated N¹⁰ emerged from these studies as the most promisingdesamino compounds for further evaluation (Jodrell et al., Proc. Amer.Assoc. Cancer Res. 31:341, 1990; Marsham et al., J. Med. Chem. 34:1594,1991).

SUMMARY OF THE INVENTION

The folate analogues of the invention are those of general structure A(illustrated below), in which C² and the associated H or methylsubstituent of the standard desamino-5,8-dideazafolic acid compound havebeen replaced by nitrogen and a lone electron pair. These changes weremade on the theory that (i) removal of H or Me might enable the moleculeto extend more deeply into the active site pocket of the enzyme, and(ii) N² might interact with active site residues by H-bonding, eitherdirectly or via a molecule of ordered water. Both of these propertieswere expected to be favorable for TS binding. Furthermore, since thecompounds of the invention have been found to be good FPGS substratesand their polyglutamates are likely to show the same increase (100-foldor more) in TS binding as that resulting from polyglutamylation of themore conventional 2-desamino from polyglutamylation of the moreconventional 2-desamino and 2-desamino-2-methylquinazolines (e.g., 3 and6), a high degree of in vivo anticancer activity is anticipated.##STR3## By "aryl moiety" is meant an aromatic monocylic or condensedpolycyclic system containing only carbon atoms in the ring itself;"heteroaryl" is an aromatic monocyclic or condensed polycyclic systemwhich has one or more heteroatoms (e.g., O, N, or S) in the ring.

In preferred embodiments, R¹ of general structure A is H, C₄ -C₄ alkyl(e.g., --CH₃ or --CH₂ CH₃), C₃ -C₄ alkenyl (e.g., --CH₂ CH═CH₂), or C₃-C₄ alkenyl (e.g., --CH₂ C═CH); R₂ may be OH, an L-α-amino acid suchL-glutamic acid or L-aspartic acid, or a peptide comprising L-α-aminoacids (such as poly-L-glutamic acid); and X is thiophene ##STR4##wherein each of Y¹ and Y², independently, is H, methyl, ethyl, or ahalogen. More preferably, Y¹ is H and Y² is H, methyl, ethyl, F, Cl, orBr (most preferably H). Specific examples of compounds of the inventioninclude: ##STR5##

The compounds of the invention are potent inhibitors of DNA synthesis inmammalian cells, such as in cultured cells or in animals (e.g., humans).By inhibiting the conversion by the enzyme TS of dUMP into dTMP, aprecursor necessary for DNA synthesis, the rate of DNA synthesis intreated cells is retarded; if the cell cannot replicate its genome, itcannot proliferate. Thus, the compound is useful for treating an animal(e.g., a mammal) having a condition, such as cancer or psoriasis,characterized by overproliferation of cells. Such a treatment would beaccomplished by introducing into the animal (e.g., by intravenousinjection, or insertion of a biodegradable implant) an amount of thecompound of the invention sufficient to reduce the rate of proliferationof the target cells. This amount would be determined in the course ofpreclinical and clinical trials of the subject compound, using standardprotocols for such trials. Guidance in this regard is provided bypublished reports concerning the dosage of other folic acid analoguespreviously tested in animals (including human patients) necessary todetect an antitumor effect (see, for example, Sessa et al., Eur. J.Cancer Clin. Oncol. 24:769-775, 1988; Calvert et al., Eur. J. Cancer16:713-722, 1980; Jackman et al., Cancer Research 50:5212-5218, 1990;Oatis and Hynes, J. Med. Chem. 20:1393-1396, 1977; and Jones et al.,Eur. J. Cancer 17:11-19, 1981, each of which publications is hereinincorporated by reference). It is expected that a useful dosage will bebetween 1.0 and 100 mg/kg, with 10-50 mg/kg being the preferred range;determination of the exact dosage will be readily determined by one ofordinary skill in the art of pharmacology. In addition, the compound ofthe invention may also be combined with another antifolate, such as thenon-polyglutamylatable dihydrofolate reductase (DHFR) inhibitortrimetrexate (cf. Galivan et al., Cancer Res. 48:2421, 1988; Galivan etal., J. Biol. Chem. 264:10685, 1989), in order to more completely blockmetabolism involving folic acid in the treated cells. Such a bivalentroute of treatment could utilize separate injections of the two agents,or could combine the two agents in a single preparation.

DETAILED DESCRIPTION

The drawings are first described.

Drawings

FIGS. 1 and 2 are representations of alternative schemes for synthesisof 10,11, and additional compounds of the invention. Described below isthe synthesis ofN-[4-[1,2,3-benzotriazin-4(3H)-on-6-yl]methylamino]benzoyl-L-glutamicacid (10, "2-aza-2-desamino-5,8-dideazafolic acid") andN-[4-[1,2,3-benzotriazin-4(3H)-on-6-yl]methyl]-N-methylamino]benzoyl-L-glutamicacid (11, "2-aza-2-desamino-N¹⁰ -methyl-5,8-dideazafolic acid"), thefirst two known members of the family embodied in general structure A,as well as methods for preparing other members of the family. Alsopresented is data showing that 10 is a potent TS inhibitor, is asubstrate for FPGS, and inhibits the growth of cultured mammalian cells.

SYNTHESIS

Schemes I and II, shown in FIG. 1 and FIG. 2, respectively, outline asynthesis of 10 and 11 and an alternative general route by whichadditional members of the series, such as those with other alkyl,alkenyl, or alkynyl groups on N¹⁰, or with a thiophene, thiazole, orpyridine ring in place of phenyl, are expected to be accessible.Sequential reactions of 5-methyl-2-nitrobenzoic acid (12) (AldrichChemical Co., Milwaukee) with thionyl chloride and methanol followed byammonia afforded 5-methyl-2-nitrobenzamide (13, 87%), which On catalytichydrogenation followed by treatment with nitrous acid (Ferrand et al.,Eur. J. Med. Chem. 22:337, 1987) yielded 2-amino-5-methylbenzylamide(14, 96%) and 6-methyl-1,2,3-benzoriazine-4(3H)-one (15, 97%),respectively (Scheme I). Improved yields in the ring closure reactionwere obtained by using less than the reported amount of acid.Confirmation of ring closure to a 1,2,3-benzotriazine-4(3H)-one camefrom the ¹ H-NMR spectrum, in which all three aromatic proton signalswere markedly deshielded (δ7.8-8.0), in agreement with the powerfulelectron-withdrawing character of the triazinone moiety. Severalattempts were made to protect N³ in 15 in the expectation thatsolubilization would be necessary if subsequent benzylic brominationwere done in CCl₄, the traditional solvent for such reactions. Treatmentof the Na salt of 15 with pivaloyloxymethyl chloride in DMF afforded asingle product which appeared to be the desired N³ -derivative 16.However, in contrast to the facile preparation of the correspondingpivaloyloxymethyl compound in the quinazoline series, all efforts torecrystallize 16 led to deacylation. We also attempted to prepare the N³-acetyl derivative 17 by reaction of the Ag salt of 15 with acetylchloride, as has been described for the analogue without a 6-methylsubstituent (Gibson et al., J. Org. Chem. 22:337, 1987). To oursurprise, the reaction of 15 yielded a product whose ¹ H-NMR spectrumcontained two Me groups, but whose UV spectrum differed from the valuesexpected from the literature (Murray et al., J. Chem. Soc. 1970:2070).Moreover, efforts to purify the product by silica gel chromatography ledonly to deacylation. Although definitive proof of its identity was notobtained, this material was tentatively assigned the O-acetyl structure18. Finally, we tried to prepare 17 from the Na salt of 15 and aceticanhydride in DMF, but no reaction occurred at room temperature and theonly product identified after heating was the benzoxazinone 19 (Murrayet al.). ##STR6##

Bromination of 15 without protection of N³ turned out to be possible byusing hot acetic acid as the solvent for the benzotrizinone. Thepresence of the brominated product 20 was evident in the ¹ H-NMRspectru, which showed a downfield singlet at δ4.63 as compared withδ3.30 for the Me group in 15. Also present were two other singlets,which we believe correspond to 21, presumably formed by solvolysis of20. Heating the crude bromination product (estimated from the ¹ H-NMRspectrum to contain roughly 40 mol % of 20) directly with dimethylN-4-aminobenzoyl-L-glutamate and NaHCO₃ in warm DMF for 3 days affordedthe protected diester 22 (45% crude yield), and further treatment of 20for a few minutes with NaOH in aqueous MeOH afforded the diacid 10(81%). The UV absorption spectrum of 10 showed a small bathochromicshift in going from acid to neutral to alkaline pH [λ_(max) (0.1M HCl)203, 224, 290 nm; λ_(max) (pH7.4) 293 nm; λ_(max) (0.1M NaOH) 298 nm. IRspectra of both 10 and 22 measured in KBr disks showed strong absorptionat 1690 cm⁻¹, indicating that the lactam tautomer 10A is present in thesolid state. The ¹ H-NMR was consistent with the benzotrizine structure,with all three aromatic ring-B protons deshielded (δ8.10) relative tothe 3',5'-protons (δ6.60) and even the 2',6'-protons (δ7.57) on thephenyl ring. However, a more interesting feature of the ¹ H-NMR spectraof diester 22 and diacid 10 was the presence of upfield signals, at δ1.7for 22 (in CDCl₃ solution) and at δ2.1 for 10 (in d₆ -DMSO solution),which we believe arise from the lactim tautomeric form 10B in thesenon-aqueous solvents. Since the peak areas for these upfield signals in10 and 22 closely approximated one proton, it appears that very littleof the lactam 10A was present under aprotic conditions. The position ofthe lactam (10A)-lactim (10B) equilibrium presumably depends not only onthe dielectric constant of the solvent, but also on its protic versusaprotic nature. ##STR7##

Biological Assays

The ability of 2-aza-2-desamino-5,8-dideazafolic acid (10) to inhibitpurified TS from L1210 murine leukemic cells (using the assay describedby Sikora et al., Biochem. Pharmacol. 37:4047, 1988, herein incorporatedby reference), to serve as a substrate for partly purified FPGS frommouse liver (using the assay described by Moran et al., Mol. Pharmacol.27:156, 1985, herein incorporated by reference), and to inhibit thegrowth of L1210 cells in culture (using the assay described by Rosowskyet al., J. Med. Chem. 34:461, 1991, herein incorporated by reference)was evaluated with the aim of comparing this compound with the analogousN¹⁰ -unsubstituted analogues of 5,8-dideazafolic acid (1) and2-desamino-5,8-dideazafolic acid (3). The results are summarized inTable 1, along with published data for N¹⁰ -propargyl-5,8-dideazafolicacid (2, CB3717).

                                      TABLE 1                                     __________________________________________________________________________    Biological Activity of 2-Aza-2-deaza-5,8-dideazafolic                         Acid (10) and Related Compounds                                                          L1210 TS.sup.b                                                                      Mouse liver FPGS.sup.c                                                                      L1210 cells.sup.d                              Compound.sup.a                                                                           K.sub.i, 1--1                                                                       K.sub.m, μM                                                                     V.sub.max (rel)                                                                    k'(rel)                                                                           IC.sub.50, μM                               __________________________________________________________________________    5,8-Dideazafolic                                                                         0.067 6.4  1.3  29  2.7                                            Acid (1)                                                                      N.sup.10 -Propargyl-5,8-                                                                 0.0027                                                                              40    0.88                                                                              2.3 3.5                                            dideazafolic Acid                                                             (CB3717, 2)                                                                   2-Desamino-5,8-di-                                                                       2.0   4.8  1.3  29  0.43                                           deazafolic Acid (3)                                                           2-Aza-2-desamino-5,8-                                                                    0.33  25   1.6  6.3 0.42.sup.e                                     dideazafolic Acid (10)                                                        __________________________________________________________________________     .sup.a With the exception of the K.sub.i for TS inhibition by 1, which is     from Sikora et al. (Biochem. Pharmacol. 37:4047, 1988), all the data for      the reference compounds 1-3 are from Jackman et al. (Cancer Res. 50:5212,     1990).                                                                        .sup.b Inhibition of purified TS from L1210 cells was determined by the       .sup.3 Hrelease assay method as described previously (Sikora et al.)          .sup.c Substrate activity for partially purified FPGS from mouse liver wa     determined as described previously (Moran et al., Mol. Pharmacol. 27:156,     1985), with relative V.sub.max and k' values expressed in comparison with     folic acid (1.0).                                                             .sup.d Cells were incubated in RPMI 1640 medium supplemented with 10%         nondialyzed fetal calf serum, and were counted after 48 h of drug             treatment.                                                                    .sup.e The IC.sub.50 for cells grown in the presence of 10 μM dThd         increased only to 0.84 μM.                                            

As shown in Table 1, a K_(i) of 0.33 μM was obtained for TS inhibitionby 10, which compared very favorably with the values of 2.0 μM reportedpreviously for the 2-desamino analogue 3 (Jackman et al., Cancer Res.50:5212, 1990). Thus, replacement of C² and the attached NH₂ group by anitrogen atom with a lone pair of electrons gave a ca. sixfold increasein TS binding. These results were consistent with our hypothesis thatthe structural change embodied in the general structure A mightfacilitate interaction with TS by allowing the A-ring to fit more snuglyinto the enzyme active site.

The K_(m) of the polyglutamates of quinazoline TS inhibitors such as 1-3to TS is known to decrease by as much as two orders of magnituderelative to the monoglutamates. It was therefore of interest todetermine whether 10 is a substrate for FPGS, since a 100-fold increasein binding could bring the K_(m) down to the low nanomolar range. Asshown in Table 1, the K_(m) of 10 for FPGS was found to be 25 μM, avalue intermediate between those of aminopterin (18 μM) (Moran et al.,Mol. Pharmacol. 27:156, 1985) and CB3717 (40 μM) (Jackman et al., CancerRes. 50:5212, 1990), but higher than those of either 1 or 3, which werein the 5-10 μM range. The relative first-order rate constant, k'(rel),calculated by dividing K_(m) (app) into the V_(max) (rel), was found tobe 6.3, a value twofold greater than that value for CB3717. However, thek' (rel) of 10 was 4.6-fold lower than the k'(rel) of 1 or 3, suggestingthat it is somewhat more efficiently polyglutamylated by the enzyme thanis CB3717, but less efficiently polyglutamylated than the two N¹⁰-unsubstituted compounds included in the comparison. However, there wasenough substrate activity to conclude that if 10 crossed the cellmembrane, it would be converted into non-effluxing polyglutamates, andthat if these polyglutamates bound tightly to TS (or other enzymes ofthe folate pathway), cell growth would be inhibited.

Incubation of L1210 cells with 10 showed that this compound was in facta potent inhibitor of growth in culture, with an IC₅₀ of 0.42 μM ascompared with 2.7 μM for 1, 3.5 μM for 2, and 0.43 μM for 3. Thus, thepotency of 10 against cultured cells was 5- to 10-fold greater than thatof either 5,8-dideazafolate or CB3717, and was comparable to that of2-desamino-5,8-dideazafolate, the lead compound whose improvedpharmacological properties relative to those of CB3717 eventually led todevelopment of the thiophene analogue 9 (Jodrell et al.; Marsham etal.).

An interesting feature of CB3717 and its 2-desamino analogues is thattheir inhibitory effect on the growth of L1210 cells is not fullyreversed by 10 μM thymidine alone, but is restored to normal levels inthe presence of either a combination of 10 μM thymidine (dThd) and 50 μMhypoxanthine (Hx) or a combination of 5 μM dThd and 5 μM folinic acid(Jackman et al., Cancer Res. 50:5212, 1990). This finding suggests thatthe intracellularly-formed polyglutamates of the quinazolines caninhibit not only TS but also DHFR, and that this results in depletion oftetrahydrofolate cofactor pools and inhibition of purine synthesis. Itwas therefore of interest to determine whether 2-aza analogues such as10 also require both dThd and HX for complete protection from theirgrowth inhibitory effect. When L1210 cells were grown in the presence of5 μM dThd, an IC₅₀ of 0.84 μM was obtained for 10, as compared with avalue of 0.42 μM when dThd was omitted from the medium. This twofolddifference is comparable to that reported for 1 and 3, but much lowerthan that reported for 2 (Jackman et al.). Thus, the pattern of growthinhibition by 10 resembles that of 1 and 3, and suggests that thiscompound may not function solely at the level of thymidylatebiosynthesis.

The potent activity of 2-aza-2-desamino-5,8-dideazafolate (10) againsttumor cells in culture, its ability to bind efficiently to TS and FPGS,and its novel molecular structure and easy synthetic access suggest thatthe compounds of the invention are readily obtainable by the methodsdisclosed herein, and will prove to be effective antifolates withmultiple enzyme targets.

EXPERIMENTAL

IR spectra were obtained on a Perkin-Elmer Model 781 double-beamrecording spectrophotometer; only peaks above 1200 cm⁻¹ are reported. UVspectra were obtained on a Varian Model 210 instrument. ¹ H-NMR spectrawere obtained on a Varian EM3460L spectrometer with Me₄ Si or Me₃Si(CH₂)₃ SO₃ Na as the reference. TLC analyses were done on fluorescentEastman 13181 silica gel sheets or Eastman 13254 cellulose sheets. Spotswere visualized under 254-nm UV illumination. Column chromatography wasdone on Baker 3405 (60-200 mesh) silica gel or Whatman DE-52 pre-swollenDEAE-cellulose. Solvents in moisture sensitive reactions were dried overLinde 4A molecular sieves (Fisher, Boston, Mass.). HPLC was done onWaters C₁₈ radial compression cartridges (analytical: 5 μm particlesize, 5×100 mm; preparative: 15 μm particle size, 25×100 mm). Meltingpoints were determined in Pyrex capillary tubes in a Mel-Temp apparatus(Cambridge Laboratory Devices, Cambridge, Mass) and are not corrected.Microanalyses were performed by Robertson Laboratory, Madison, N.J.

6-Methyl-1,2,3-benzotriazin-4(3H)-one (15). A mixture of5-methyl-2-nitrobenzoic acid (12) (36.2 g, 0.2 mol) and SOCl₂ (50 mL)was heated under reflux for 20 min, during which a homogeneous solutionwas obtained. After removal of the excess SOCl₂ with the aid of a wateraspirator, the residue was dissolved in dry THF (40 mL) and the solutionadded dropwise with stirring to an ice-cold solution of NaOH (8 g, 0.2mol) in 28% NH₄ OH (300 mL). The precipitate was collected, washed withwater, and recrystallized from EtOH-H₂ O to obtain5-methyl-2-nitrobenzamide (13) (31.5 g, 87%) as a white solid; mp176°-177° C. [lit. (Findeklee, Ber. 38:3558, 1905) mp 176°-177° C.]; IR(KBr) V 1655 Cm⁻¹ (amide C═O). A solution of 13 (31.5 g, 0.175 mol) inMeOH (250 mL) was shaken With 5% Pd-C (0.5 g) under 3 atm. of H₂ for 24h in a Parr apparatus. A solid formed after the initial heat of reactionsubsided. The mixture was heated to boiling to redissolve the product,and was filtered while hot. The filtrate was evaporated under reducedpressure and the residue dried at 90° C. for 1 h (caution: somesublimation may occur) to obtain 2-amino-5-methylbenzamide (14) as awhite solid (25.3 g, 96%); mp 173°-175° C. [lit. (Findeklee) mp 179°C.]; ¹ H-NMR (CDCl₃) δ6.60 (d, 1H, J=8 Hz, C₃ -H). To an ice-coldsuspension of 14 (25.3 g, 0.168 mol) in 3.6N HCl (260 mL) was addeddropwise over 25 min a solution of NaNO₂ (12.75 g) in H₂ O (100 mL)while keeping the internal temperature below 5° C. After another 20 minof stirring at this temperature, 10N NaOH (100 mL) was added, causingall the solid to dissolve. The solution was acidified to pH 2 with 12NHCl and chilled. The solid was filtered and recrystallized from EtOH toobtain off-white needles (26.2 g, 97%); mp 217°-218° C. (dec, gasevolution) [lit. (Ferrand et al., Eur. J. Med. Chem. 2:337, 1987) mp219°-220° C.]; IR (KBr) v 1680 (lactam C═O); ¹ H--NMR (d₆ --DMSO+D₂ O)δ3.30 (s, 3H, 6--Me), 8.00 (m, 3H, aryl); UV (95% EtOH) λ_(max) 208,225, 282 nm.

Dimethyl N-[4-(1,2,3-Benzotriazin-4(3H)-on-6-yl)methyl]aminobenzoyl-L-glutamate (22). N-Bromosuccinimide (1.37 g, 7.7mmol) was added in a single portion to a solution of 14 (1.13 g, 7 mmol)in glacial AcOH (70 mL) in an oil bath at 60° C. The resulting solutionwas heated at 70° C. with illumination from a 150-watt floodlamp(General Electric) for 2 h. After evaporation of the solvent underreduced pressure, the residue was partitioned between CHCl₃ and H₂ O.The CHCl₃ layer was washed with 5% NaHCO₃, rinsed with H₂ O, andevaporated to obtain a product (0.946 g) which was estimated to consistof a 2:3 mixture of 6-bromomethyl-1,2,3-benzotriazin-4(3H)-one (20) [¹H--NMR (CDCl₃) δ4.63] and unchanged 15 [¹ H--NMR (CDCl₃) δ3.30]. Theentire mixture of 20 and 15 from the bromination reaction (estimated tocontain 1.97 mmol of 20 from the δ4.63/δ3.30 ratio) was added in asingle portion to a solution of dimethyl N-(4-aminobenzoyl)-L-glutamate(0.588 g, 2 mmol) (Koehler et al., J. Amer. Chem. Soc., 80:5779, 1958)in dry DMF (10 mL). Then, solid NaHCO₃ (0.168 g, 2 mmol) was added andthe mixture was kept in an oil bath at 65° C. for 3 days. The solventwas evaporated under reduced pressure and the residue partitionedbetween CHCl₃ and H₂ O. The CHCl₃ layer (TLC: R_(f) 0.70, 0.65, 0.4,0.0; silica gel, 19:1 CHCl₃ -MeOH) was evaporated and the residuechromatographed on a silica gel column (45 g, 2.5×40 cm) with 20:1 CHCl₃--MeOH as the eluent. Pooled fractions containing the spot with R_(f)=0.4 were evaporated in two batches, and the residues were driedseparately in vacuo (P₂ O₅, 65° C.). The first batch (0.254 g, 28%) wasTLC-homogeneous, whereas the other (0.154 g, 17%) contained a smallimpurity. Rechromatography of the TLC-homogeneous batch affordedanalytically pure 10 as a beige powder (0.22 g, 87% recovery)); mp94°-102° C.; IR (KBr) v 3430, 3040, 2960, 1740, 1690, 1640, 1615, 1580,1515, 1445, 1420, 1335, 1285, 1265 cm⁻¹ ; NMR (CDCl₃) δ1.71 (br s, 1H,lactim OH), 2.43 (m, 4H, CH₂ CH₂), 3.63 (s, 3H ₆₅ -COOMe), 3.75 (s, 3H,α-COOMe), 4.65 (m, 4H, CH₂ N, NH, α-CH), 6.58 (d, J=9 Hz, 2H, 3'- and5'-H), 7.68 (d, J=9 Hz, 2H, 2'- and 6'- H), 7.8-8.4 (m, 3H, aryl). Anal.Calcd for C₂₂ H₂₃ N₅ O₆.0.6H₂ O: C, 56.92; H, 5.25; N, 15.08. Found: C,57.05; H, 5.00; N, 14.71.

N-[4-(1,2,3-Benzotriazin-4(3H)-on-6-yl)methyl] aminobenzoyl-L-glutamicAcid (10). A stirred cloudy solution of 22 (195 mg, 0.42 mmol) in MeOH(5 mL) was treated dropwise with 1N NaOH (5 mL) over 1 min. The solutionquickly became homogeneous, and after another 5 min the pH wa adjustedto neutrality with HCl. The MeOH was evaporated under reduced pressureand the product purified by preparative HPLC (8% MeCN in 0.05M NH₄ OAc,pH 6.9). Pooled pure fractions were concentrated by rotary evaporationand then freeze-dried. The residue was redissolved in H₂ O (15 mL) andthe solution lyophilized again to obtain a pale-yellow solid (162 mg,81%); mp ca. 150° C. (gas evolution after softening at 140°-145° C.); IR(KBr) v 3420, 3150, 3040, 2980, 1690, 1615, 1580, 1555, 1520, 1445,1405, 1330, 1285, 1270 cm⁻¹ ; NMR (d₆ -DMSO) δ2.10 (m, 5 H, CH₂ CH₂,lactim OH), 4.15 (m, 1H, α-CH), 4.57 (m, 2H, CH₂ N), 6.60 (d, J=8 Hz,3'- and 5'-H), 7.02 (m, 1H, NH), 7.57 (d, J=8 Hz, 2H, 2'- and 6'-H, withoverlapped m, 1H, NH), 8.10 (m, 3H, aryl); UV (0.1M HCl) λ_(max) 203 nm(ε27,700), 224 (27,600), 290 (12,300); λ_(max) (pH 7.4 phosphate buffer)216-218 nm (plateau, ε32,200), 293 (24,300); λ_(max) (0.1M NaOH) 298 nm(ε25,300). Anal. Calcd for C₂₀ H₁₉ N₅ O₆.0.75 NH₃.2H₂ O: C, 50.66; H,5.37; N, 16.98. Found: C, 50.40; H, 5.41; N, 16.80.

Synthesis of Other 2-Aza-2-desamino-5,8-dideazafolic Acid Analogues(General Procedure). A solution of 15 in glacial AcOH (ca. 50 mL/g) istreated with a single portion of N-bromosuccinimide (10% molar excess)and heated at 75° C. under a 75-watt lamp for 2 h. The reaction mixtureis evaporated to dryness under reduced pressure, the residue ispartitioned between CHCl₃ and 5% NaHCO₃, and the organic layer isevaporated. The resulting solid, consisting of a mixture of unreacted 15and bromide 20, is dissolved directly in dry DMF (ca. 10 mL/g) andtreated with an amount of the appropriate N-aroyl-L-glutamate diester(prepared, for example, as described by Jones et al., Eur. J. Cancer17:11, 1981; and/or Marsham et al.) equimolar to that of bromide 20estimated to be present in the crude bromination produce by analysis ofthe ¹ H--NMR spectru. Solid NaHCO₃ (50% excess) is also added, and themixture is stirred at 65° C. for 70 h. The DMF is evaporated underreduced pressure, and the residue is partitioned between CHCl₃ andwater, with enough glacial AcOH added to bring the pH to <6. The CHCl₃layer is evaporated, and the product is chromatographed on a column ofsilica gel (40 g) which is eluted first with CHCl₃ followed by mixturesof CHCl₃ and MeOH (up to 10% as needed). Fractions are monitored by TLC(silica gel, CHCl₃ or CHCl₃ --MeOH as needed), and appropriate fractionsare pooled and evaporated. The residue, consisting of the diethyl esterof the 2-aza-2-desamino-5,8-dideazafolate analogue, is suspended in 50%EtOH, and a stoichiometric amount of 1N NaOH is added in an equal volumeof water. After 15 min at room temperature, the mixture is filtered andthe filtrate adjusted to pH 8 by careful dropwise addition of 1N HCl.The EtOH is evaporated under reduced pressure, any insoluble materialpresent is removed by filtration, and the filtrate is placed on aDEAE-cellulose column (40 g, HCO₃ -form). The column is washed firstwith water to remove salts and any residual benzotriazinone 15, and thenwith 0.4M NH₄ HCO₃. Appropriate fractions are pooled and subjected torepeated freeze-drying to obtain the final product, typically as ahydrated ammonium salt.

Other compounds of the invention may be prepared by appropriatevariations in the above procedure. For example, the moiety attached tothe 6-position of the benzotriazinone can be varied by substituting anappropriate reagent for the N-aroyl-L-glutamate diester utilized above;preparation of such a reagent would be readily accomplished by asynthetic organic chemist of ordinary skil, utilizing procedures such asthose described in Jones et al., j. Med. Chem. 29:1114, 1986; Jones etal., Eur. J. Cancer 17:11, 1981; and Marsham et al.

Other embodiments are within the following claims.

What is claimed is:
 1. A compound having the formula ##STR8## wherein Xis thiophene, thiazole, pyridine, phenyl, or phenyl substituted withmethyl, ethyl, or halogen;R¹ is H, C₁ -C₄ alkyl, C₃ -C₄ alkenyl, or C₃-C₄ alkynyl; and R² is OH, L-glutamic acid or L-aspartic acid.
 2. Thecompound of claim 1, wherein X is thiophene, thiazole, pyridine, or##STR9## wherein each of Y¹ and Y², independently, is H, methyl, ethyl,or a halogen.
 3. The compound of claim 2, wherein said Y¹ is H and saidY² is H, methyl, ethyl, F, Cl, or Br.
 4. The compound of claim 2,wherein said X is ##STR10##
 5. The compound of claim 1, wherein said R¹is H, --CH₃, or --CH₂ C.tbd.CH.
 6. The compound of claim 1, wherein saidcompound has the formula ##STR11##
 7. The compound of claim 1, whereinsaid compound has the formula ##STR12##
 8. The compound of claim 1,wherein said compound has the formula ##STR13##
 9. The compound of claim1, wherein said compound has the formula ##STR14##
 10. The compound ofclaim 1, wherein said compound has the formula ##STR15##
 11. Atherapeutic composition comprising an effective amount of the compoundof claim 1 in a pharmaceutically-acceptable carrier.